Cytoreductive chemotherapy in induction therapy plays a key role in the prognosis of patients with low‐risk acute promyelocytic leukaemia

Abstract In order to explore the risk factors of relapse and potential optimized therapeutic regimen of low‐risk acute promyelocytic leukaemia (APL), here we retrospectively analysed 282 patients who were diagnosed between February 2014 and September 2021. The median follow‐up was 59 (9–102) months. The 5‐year overall survival and cumulative relapse incidence were 97.9% and 5.9%, respectively. In terms of different cytoreductive therapies, 86 patients were administered with hydroxycarbamide (30.5%), 113 with anthracyclines or cytarabine (40.1%), 31 with etoposide (11.0%) and 52 with no cytoreductive therapy (18.4%) during the induction therapy. The hydroxycarbamide treatment group did not decrease the relapse rate compared to the no cytoreduction group (11.4% vs. 5.9%, p = 0.289). Compared with the hydroxycarbamide group, the anthracyclines/cytarabine treatment group showed improved 5‐year RFS (88.145% vs. 98.113%, p = 0.008). Multivariate Cox regression analysis revealed that myeloblasts in bone marrow at diagnosis, and PML‐RARA transcript level of 6.5% or more after induction therapy were associated with a subsequent risk of relapse. The only factor positively reducing the relapse rate was anthracyclines/cytarabine cytoreductive treatment. In conclusion, cytoreductive chemotherapy in induction therapy plays a potential key role in the prognosis of low‐risk APL.


| INTRODUC TI ON
Despite the success of low-risk acute promyelocytic leukaemia (APL) in the all-trans retinoic acid (ATRA) plus arsenicals era, several important clinical issues continue to account for treatment failure including early death (ED) and disease relapse.2][3][4][5][6] Differentiation syndrome (DS), which is a common side-effect of differentiating agents during induction treatment, occurred in 11%-28% of patients with low-risk APL. 2,7,8veral studies demonstrated that leukocytosis increases susceptibility of DS and even ED. 9Approximately 70% of low-risk patients treated with arsenicals develop leukocytosis with induction therapy, with a median peak white blood cell (WBC) count of 20 × 10 9 /L at about 10 days from the start of treatment. 7Thus, the efficient and safe reduction of WBC count by cytoreductive therapy is the key point in the induction therapy of low-risk APL.Guidelines from the National Comprehensive Cancer Network (NCCN) recommended hydroxyurea, anthracycline or gemtuzumab ozogamicin (GO) for cytoreduction. 10Limited effectiveness of hydroxyurea, inconvenient intravenous injection of anthracycline or unavailability of GO in China hampered the progress of the therapeutic regimens for APL.
The combination of ATRA and ATO resulted in statistically significant better EFS and OS rates, reduced cumulative incidence of relapse and lower toxicity, compared to ATRA plus chemotherapy. 2,3ile previous studies by our group and others showed a relapse of 1.0%-4.8%for low-risk APL, and the median time to haematological relapse was 20.5 months after a haematological complete remission (CR). 2,5We and others have indicated that the addition of cytarabine in induction therapy might correlate with a lower relapse rate. 5,11,12ether cytoreduction in induction therapy in the setting of ATRA plus arsenicals has prognostic significance in APL, besides its role in leukocytosis, remains unclear.
Etoposide is an antitumour inhibitor of topoisomerase II widely used in the treatment of several malignant haematological tumours.
The successful experience in two high-risk APL patients demonstrated the efficacy, safety and convenience of oral etoposide as an alternative cytoreductive agent at the initial stage of induction therapy. 13Therefore, the present study was conducted to explore the potential role of cytoreduction during induction therapy on prognosis, and further exploit the all-oral induction regimen for low-risk APL with etoposide combined with ATRA plus realgar-indigo naturalis formula (RIF), an oral realgar (As₄S₄)-containing formula, as the front-line therapy for low-risk APL.

| Patients
All consecutive low-risk APL patients who received ATRA plus arsenic for induction and consolidation therapy at the Peking University People's Hospital, Peking University Institute of Haematology, between February 2014 and September 2021, were analysed.Low-risk patients were defined as a WBC count of no more than 10 × 10 9 /L at diagnosis. 10Between February 2014 and August 2015, low-risk APL patients randomly received RIF-ATRA or arsenic trioxide (ATO)-ATRA for induction and consolidation therapy as part of a randomized, controlled phase III clinical trial (ChiCTR-TRC-13004054) and data of 59 patients was reported in our previous study. 4Between September 2015 and March 2018, low-risk APL patients were treated with intravenous or oral arsenic based on patients' choice and data from 153 patients was reported in our previous study. 5After April 2018, low-risk APL patients were treated with RIF plus ATRA as a front-line therapy recommended by the 'Chinese Guidelines for Diagnosis and Treatment of Acute Promyelocytic Leukemia (2018)'. 14Approval was obtained from the Ethics Committee of Peking University People's Hospital (No.2022PHB342-001), and written informed consent was obtained from all patients according to the Declaration of Helsinki.Cytoreduction therapy, including hydroxycarbamide, anthracyclines and cytarabine during induction therapy was administrated based on the 2018 Chinese APL guidelines. 14Given the unavailability and inconveniences of intravenous chemotherapy, our centre explored the routine of oral etoposide as cytoreductive therapy from January 2020.

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Consolidation therapy was conducted in accordance with the 2018 Chinese APL guidelines.We considered two cycles of ATRA and one cycle of RIF or ATO to be one complete round of consolidation therapy, and thus, the patients received a total of approximately four rounds of consolidation therapy.

| Definitions of outcomes
Leukocytosis was defined as a white blood cell (WBC) count greater than 10 × 10 9 /L during induction therapy.Haematological CR was defined as a proportion of BM blasts less than 5%, no blasts in Auer rods, no extramedullary disease, an absolute neutrophil count greater than 1 × 10 9 /L and a platelet count greater than 100 × 10 9 /L, with no red-cell transfusions.Haematological relapse was defined as the recurrence of blasts greater than 5% in the BM, the reappearance of blasts in the blood or the development of extramedullary disease infiltrates in any Overall survival (OS) was defined as the time from diagnosis to the date of death, regardless of any cause or last follow-up.Relapse-free survival (RFS) was defined as the time from CR to relapse (including hematologic and molecular relapses) or death.In the PETHEMA studies, DS severity was graded according to the number of clinical criteria met to predict outcomes.Patients with four or more of the above signs and symptoms were classified as having severe DS, whereas those with less than four signs and symptoms were classified as having moderate DS. 15,16Acute and subacute toxicity of anticancer drugs was graded according to the WHO classification standard.During induction therapy and consolidation therapy, common haematological and non-haematological adverse events were monitored twice per week.

| Response evaluation and molecular monitoring
Every bone marrow sample in our institute was tested and reviewed by two independent hematopathologists.Bone marrow (BM) was evaluated for induction therapy at 4-6 weeks after the blood cell count recovered, namely, when the neutrophil absolute count was ≥1.0 × 10 9 /L and the platelet count was ≥100 × 10 9 /L.BM assessments were conducted every 2 months during consolidation therapy.
Following consolidation therapy, BM assessment, including morphology, immunophenotyping and PML-RARA transcript detection, was performed every 3 months during the first 2 years and every 6 to 12 months thereafter.Quantitative RT-PCR was performed to monitor the levels of PML-RARA transcript in BM as previously described. 5Definitions of haematological CR, haematological relapse, complete molecular remission (CMR), molecular relapse, overall survival (OS), relapse-free survival (RFS) and toxic effects are explained as previously described. 5

| Treatment response
After induction therapy, all 276 evaluable patients (100%) achieved haematological CR except for six patients who died early.Of the 282 patients evaluable for OS, the 5-year OS was 97.9% (Figure 2A).Of the 267 patients evaluable for relapse, 14 patients (5.2%) relapsed throughout follow-up, with a 5-year cumulative relapse incidence of 5.9% (Figure 2B).Among them, 12 patients experienced haematological relapse after CMR, one of whom developed central nervous system leukaemia (CNSL).The remaining two patients experienced molecular relapse, one of whom developed CNSL.The median time to relapse, including haematological, molecular and extramedullary relapse, was 18.5 (IQR 12.25-24.5,range 6.2-52.0)months after haematological CR.No death occurred in patients with relapse during the follow-up.

| Cytoreduction therapy during the induction therapy
In terms of different cytoreduction therapies, 86 patients were administered with hydroxycarbamide (30.5%), 113 with anthracyclines or cytarabine (40.1%), 31 with etoposide (11.0%) and 52 with no cytoreductive therapy (18.4%) during induction therapy.Two  1 and 2).There were no significant differences in the incidences of CR and CMR after consolidation therapy, or the time to CR after induction therapy among the groups (Tables 1 and 2).No difference was observed in the incidence of ED among the four groups (Table 1).Our previous study found that a PML-RARA transcript level of 6.5% or more at the end of induction therapy was associated with a subsequent risk of relapse. 5Compared to patients with no cytoreduction therapy or only hydroxycarbamide during induction therapy, the incidence of PML-RARA transcript levels of ≥6.5% was significantly decreased at the end of induction therapy in those who received anthracyclines/cytarabine and etoposide (27.5% vs. 26.2% vs. 15.5% vs. 0%, p = 0.003, Table 1).Notably, the hydroxycarbamide treatment group did not decrease the relapse rate compared to the no cytoreduction group (11.4% vs. 5.9%, neither nor the 5-year RFS (88.145% vs. 93.752%,p = 0.292).Compared with the hydroxycarbamide group, the anthracyclines/cytarabine treatment group showed improved 5-year RFS (88.145% vs. 98.113%, p = 0.008, Figure 2C).Data from the etoposide treatment group was reported in the Supplementary Results because of the short follow-up time (median 17.0 months, range 9.0-24.0months).

| Adverse events
The main adverse effects are listed in Table 4. Adverse effects mainly consisted of neutropenia, thrombocytopenia, liver injury and infection.Forty-two patients (14.9%) had Grade 1-4 diarrhoea.Sixteen (5.6%) patients in the ATRA-RIF group were switched to ATRA-ATO due to Grade 3-4 diarrhoea or hemafecia.In total, 183 patients (64.9%) had Grade 1-4 liver injury.Grade 3-4 liver injury was reported in 30 (11.2%) of 267 patients and the toxic effects resolved with temporary discontinuation of arsenic, ATRA or both.

| DISCUSS ION
This study sheds light on the crucial role of cytoreductive chemotherapy during induction therapy in the prognosis of low-risk APL in the ATRA plus arsenicals era.APL has become curable under ATRA and arsenicals in low-risk patients. 17However, there are thorny issues, such as ED and relapse, which are yet to be addressed.After administration of ATRA and arsenicals, the WBC TA B L E 2 Relapse of different cytoreduction during induction therapy.The time to haematological/molecular/extramedullary relapse after a haematological CR. c p values of CMR and relapse were among the four groups.p-values of relapse, 2-year cumulative relapse, 2-year relapse-free survival, time to relapse and follow-up time were among three groups of no cytoreduction group, the hydroxycarbamide treatment group and the anthracyclines/cytarabine treatment group.
count will typically increase and may lead to DS, which is one of the main reasons of ED and occurs in 11%-28% of patients with low-risk APL.Chemotherapy-associated secondary myeloid malignancies and more early and late adverse events need long-term observation.
This study explored an all-oral regimen of etoposide as cytoreductive therapy combined with RIF and ATRA for low-risk APL.
NCCN guidelines recommend anthracycline-based chemotherapy as one part of induction therapy for newly diagnosed APL to reduce WBC count.However, the guidelines do not elaborate on when to initiate chemotherapy.Similarly, there is a lack of studies on the optimal timing of chemotherapy during induction treatment. 25de Botton et al suggested that early addition of chemotherapy with low WBC counts significantly reduced the incidence of ATRA syndrome during induction therapy. 26Here we recommended that oral etoposide, with a relatively slow effect on cytoreduction, was initiated to reduce tumour load as early as possible in order to reduce the rate Treatment procedure 4,14 All-trans retinoic acid (25 mg/m 2 /day divided into two oral doses) was administered immediately upon suspicion of APL based on morphological features.RIF (60 mg/kg/day divided into three oral doses) or ATO (one intravenous dose of 0.15 mg/kg/day) was administered upon detection of the PML-RARA fusion gene via reversetranscription polymerase chain reaction (RT-PCR) or the t(15;17) translocation via conventional karyotyping or fluorescence in situ hybridization.Since genetic confirmation of the diagnosis takes 5 to 7 days, the arsenic therapy was administered approximately 5 to 7 days later than ATRA.RIF or ATO plus ATRA was maintained until CR was reached after induction therapy.
site.Molecular relapse was defined as a confirmed reversal of PML-RARA positivity within 4 weeks.Complete molecular remission (CMR) was defined as the absence of detectable PML-RARA transcripts by quantitative RT-PCR.The sensitivity level of RT-PCR for PML-RARA transcriptions, as evaluated by series dilution experiments, was 10 −4 .
Kaplan-Meier was used to estimate relapse rates and OS.Log-rank tests were performed using the Statistical Package for the Social Sciences (SPSS), version 22.0 (SPSS, Inc., Chicago, IL, USA).A p < 0.05 was considered statistically significant.Exploration of factors associated with relapse was based on the Cox proportional hazards regression model with R software (version 4.2.0).Risk factors with a p < 0.2 in the univariate analysis were selected for further evaluation by multivariate Cox proportional hazard models.Factor leukocytosis was not selected because of its collinearity with the factor of anthracyclines/ cytarabine.Patients An overview of the study algorithm can be found in Figure 1.From February 2014 to September 2021, 282 patients were diagnosed with low-risk APL and received ATRA plus arsenic as an induction and consolidation therapy.A total of 246 patients (87.2%) received RIF and ATRA as front-line induction therapy, and 36 (12.8%) received ATO and ATRA.During induction therapy, six patients died early.Nine patients were withdrawn after induction therapy.Finally, a total of 267 patients were evaluated for relapse.Follow-up was last conducted on 31 August 2022.The median follow-up was 59 months [interquartile range (IQR), 41-81; range 9-102].A summary of the baseline characteristics of the patients can be found in Table The median (range) time to haematological CR was 36 (28-49) days.Leukocytosis occurred in 165 (58.5%) of the 282 patients during induction therapy.Moreover, the WBC count increased F I G U R E 1 Study diagram.CR, complete remission.above 4 × 10 9 /L during induction therapy in almost all the patients (263/282, 93.3%).Overall, 49 patients (49/282, 17.4%) developed DS, including moderate and severe forms.Six patients developed ED: three patients died of severe DS, two of cerebral haemorrhage on days 7 and 14, respectively, and one of intracranial infection on day 29 of induction therapy.No patient died during the consolidation therapy.
patients in the no cytoreduction group developed leukocytosis during induction with the maximum WBC count of 13.22 × 10 9 /L and back to less than 10 × 10 9 /L within 1 day without any cytoreduction therapy.Hydroxycarbamide was initiated in patients with a WBC count of 4.31-10.45× 10 9 /L during induction.Leukocytosis during induction occurred in 43 (50.0%)patients with the maximum WBC count of 17.36 × 10 9 /L.Anthracyclines or cytarabine was initiated in patients with a WBC count of 9.18-13.69× 10 9 /L during induction; leukocytosis during induction occurred in 100 (88.5%) patients with a maximum WBC count of 35.79 × 10 9 /L.Except for three patients developed early death in this group, 75 patients received cytarabine alone, 7 received mitoxantrone alone, 7 received daunorubicin alone, 8 received cytarabine and mitoxantrone, 13 received cytarabine and daunorubicin.Thirty-three of 110 patients received hydroxycarbamide with a mean cumulative dose of less than 7.0 g before the initiation of anthracyclines/cytarabine. Etoposide was initiated in patients with a WBC count of 4.21-9.82× 10 9 /L during induction.Details about cytoreduction therapies can be found in Supplementary Results.In order to illustrate the role of cytoreductive therapy in the prognosis of low-risk APL patients, indicators of treatment response, ED and relapse were analysed among the groups (Tables

F I G U R E 2 | 5 of 10 ZHU
Abbreviations: CMR, complete molecular remission; CR, complete remission; ED, early death.PML-RARA, promyelocytic leukaemia retinoic acid receptor alpha.a CR after induction therapy .
Abbreviations: CMR, complete molecular remission; CR, complete remission.a CMR after the fourth round of consolidation therapy. b

F I G U R E 3
Multivariate analysis of relapse.CI, confidence interval; HR, hazard ratio; PML-RARA, promyelocytic leukaemia retinoic acid receptor alpha.TA B L E 4Adverse events reported in treatment groups.
2,4,7Our study revealed 58.5% Leukocytosis during induction, 17.4% DS, and 2.1% ED.Cytoreductive chemotherapy may partially prevent or control the risks of DS and still plays a vital role in induction therapy.Univariate analysis of relapse.Myeloblasts in bone marrow at diagnosis, PML-RARA of 6.5% or more and anthracyclines/cytarabine cytoreductive treatment were associated with a subsequent risk of relapse.According to our Cox proportional hazard regression model, cytoreductive chemotherapy, which had the most weight, was shown to reduce the risk of relapse.
ratio; PML-RARA, promyelocytic leukaemia retinoic acid receptor alpha; PRAME, preferentially expressed antigen in melanoma; RFS, relapse-free survival; WT1, Wilms tumour 1.In the study, patients who received anthracyclines or cytarabine as cytoreduction had a decreased rate of PML-RARA of 6.5% or more at the end of induction therapy, and improved RFS compared to patients with hydroxycarbamide during induction therapy.To our knowledge, research on ATRA-arsenicals ± cytoreductive therapy is not available to date.Conclusions about the role of chemotherapy in the induction therapy of APL were drawn from studies about ATRA + chemotherapy.That's why the aim of our study is to explore the potential role of cytoreduction during induction therapy in the ATRA plus arsenicals era.In our cohort, 14 patients (5.2%) relapsed during follow-up, with a 5-year cumulative relapse incidence of 5.9%.